Human parvovirus B19 is a member of the family Parvoviridae, genus Erythrovirus and is a small 22-nm icosahedral nonenveloped virus with a linear single-stranded DNA molecule of approximately 5,600 nucleotides. The viral genome encodes three major proteins, VP1, VP2 and NS1. See, Shade et al., J. Virol. (1986) 58:921-936 and FIG. 1 herein. VP1 (83 kDa) and VP2 (58 kDa) are the structural proteins of the capsid. The two proteins are encoded in overlapping reading frames from about nucleotides 2444 to 4789 and about 3125 to 4789, respectively. VP2 constitutes 95% of the capsid and the larger VP1 protein only 5% of the capsid. VP1 is required for the mature conformation of the virus. NS1 (77 kDa), is a nonstructural protein and is present only in the nuclear fraction of infected cells and absent from the cytoplasm and intact virions in sera.
Parvovirus B19 was first discovered in the sera of normal blood donors and is the only member of the family Parvoviridae known to be pathogenic in humans. The virus is associated with a wide range of disease manifestations. Human parvovirus B19 normally causes an asymptomatic or mild self-limiting infection in children. In adults, parvovirus B19 may cause a rash, transient symmetrical polyarthralgia and arthritis. Parvovirus B19 has been associated with transient aplastic crisis (TAC) in patients with underlying hemolytic disorders. Chronic B19 infection and persistent anemia have been reported in immunocompromised patients with acute leukemia, congenital immunodeficiencies, AIDS, and following bone marrow transplantation. Parvovirus B19 has also been associated with fetal death in pregnant women.
In most countries, B19 virus infection generally occurs during childhood, with approximately 50% of children having anti-B19 antibodies by the age of 15 years. B19 antibody prevalence may further increase during lifetime and reaches values higher than 90% in elderly individuals.
In human parvovirus B19 infection, initial viral replication is believed to occur in the respiratory tract. The virus then targets cells in the bone marrow. This leads to large-scale viral replication with reported viremia of between 102 to 1014 particles/ml, occurring 7-10 days after infection but prior to the onset of symptoms. Cessation of viremia coincides with the detection of specific IgM antibodies that remain elevated for two to three months. Anti-B19 IgG antibodies are detected a few days after IgM antibodies appear and persist lifelong.
The absence of a lipid envelope and limited DNA content make parvovirus B19 extremely resistant to physicochemical inactivation. Parvovirus B19, especially at high concentration, can withstand conventional heat treatment of blood products and transmission of B19 through the administration of solvent-detergent-treated factor VIII and steam- or dry-heated factor VIII and IX preparations has been documented.
Human parvovirus B19 cannot be grown in conventional cell cultures making laboratory detection and isolation of the virus extremely difficult. Thus, for many years, the only source of antigen consisted of sera from viremic patients. Recombinant antigens have been produced for use in serological assays in an attempt to circumvent these problems. See, e.g., Sisk and Berman, Biotechnology (1987) 5:1077-1080; U.S. Pat. No. 6,204,044. Immunoenzymatic IgM capture assays have been used to detect anti-B19 IgM, as well as to diagnose recent B19 infection. The diagnostic performance of a number of commercially available tests, however, is not homogenous. In addition, IgM-based diagnostic tests cannot detect the virus during the viremic stage of infection and once IgM antibodies are synthesized, they can remain in circulation for several months after the end of viremia.
The high prevalence of B19 antibodies in the normal population together with the fact that high viremia usually persists for only one week, make the use of serological based tests impractical. In addition, in immunocompromised patients, serological diagnosis may be unreliable.
Nucleic acid-based hybridization assays, such as dot blot and in situ hybridization have been used for B19 detection. These assays generally have detection limits of 1 to 0.1 pg viral DNA (˜104-105 viral particles). PCR has greater sensitivity (˜100 genome copies). However, DNA hybridization techniques are time consuming and limited in use and PCR is impractical for screening large numbers of samples.
Therefore, there remains a need for the development of reliable diagnostic tests to detect parvovirus B19 in viremic samples, in order to prevent transmission of the virus through blood and plasma derivatives or by close personal contact.